EGFR signaling and pharmacology in oncology revealed with innovative BRET-based biosensors.

Mutations of receptor tyrosine kinases (RTKs) are associated with the development of many cancers by modifying receptor signaling and contributing to drug resistance in clinical settings. We present enhanced bystander bioluminescence resonance energy transfer-based biosensors providing new insights into RTK biology and pharmacology critical for the development of more effective RTK-targeting drugs. Distinct SH2-specific effector biosensors allow for real-time and spatiotemporal monitoring of signal transduction pathways engaged upon RTK activation. Using EGFR as a model, we demonstrate the capacity of these biosensors to differentiate unique signaling signatures, with EGF and Epiregulin ligands displaying differences in efficacy, potency, and responses within different cellular compartments. We further demonstrate that EGFR single point mutations found in Glioblastoma or non-small cell lung cancer, impact the constitutive activity of EGFR and response to tyrosine kinase inhibitor. The BRET-based biosensors are compatible with microscopy, and more importantly characterize the next generation of therapeutics directed against RTKs.

Effector membrane translocation biosensors reveal G protein and ßarrestin coupling profiles of 100 therapeutically relevant GPCRs.

The recognition that individual GPCRs can activate multiple signaling pathways has raised the possibility of developing drugs selectively targeting therapeutically relevant ones. This requires tools to determine which G proteins and ßarrestins are activated by a given receptor. Here, we present a set of BRET sensors monitoring the activation of the 12 G protein subtypes based on the translocation of their effectors to the plasma membrane (EMTA). Unlike most of the existing detection systems, EMTA does not require modification of receptors or G proteins (except for Gs). EMTA was found to be suitable for the detection of constitutive activity, inverse agonism, biased signaling and polypharmacology. Profiling of 100 therapeutically relevant human GPCRs resulted in 1500 pathway-specific concentration-response curves and revealed a great diversity of coupling profiles ranging from exquisite selectivity to broad promiscuity. Overall, this work describes unique resources for studying the complexities underlying GPCR signaling and pharmacology.

Extracellular succinate hyperpolarizes M2 macrophages through SUCNR1/GPR91-mediated Gq signaling.

Succinate functions both as a classical TCA cycle metabolite and an extracellular metabolic stress signal sensed by the mainly Gi-coupled succinate receptor SUCNR1. In the present study, we characterize and compare effects and signaling pathways activated by succinate and both classes of non-metabolite SUCNR1 agonists. By use of specific receptor and pathway inhibitors, rescue in G-protein-depleted cells and monitoring of receptor G protein activation by BRET, we identify Gq rather than Gi signaling to be responsible for SUCNR1-mediated effects on basic transcriptional regulation. Importantly, in primary human M2 macrophages, in which SUCNR1 is highly expressed, we demonstrate that physiological concentrations of extracellular succinate act through SUCNR1-activated Gq signaling to efficiently regulate transcription of immune function genes in a manner that hyperpolarizes their M2 versus M1 phenotype. Thus, sensing of stress-induced extracellular succinate by SUCNR1 is an important transcriptional regulator in human M2 macrophages through Gq signaling.

GPCR-G Protein-ß-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Quantification of ligand bias for clinically relevant ß2-adrenergic receptor ligands: implications for drug taxonomy.

The concepts of functional selectivity and ligand bias are becoming increasingly appreciated in modern drug discovery programs, necessitating more informed approaches to compound classification and, ultimately, therapeutic candidate selection. Using the ß2-adrenergic receptor as a model, we present a proof of concept study that assessed the bias of 19 ß-adrenergic ligands, including many clinically used compounds, across four pathways [cAMP production, extracellular signal-regulated kinase 1/2 (ERK1/2) activation, calcium mobilization, and receptor endocytosis] in the same cell background (human embryonic kidney 293S cells). Efficacy-based clustering placed the ligands into five distinct groups with respect to signaling signatures. In some cases, apparent functional selectivity originated from off-target effects on other endogenously expressed adrenergic receptors, highlighting the importance of thoroughly assessing selectivity of the responses before concluding receptor-specific ligand-biased signaling. Eliminating the nonselective compounds did not change the clustering of the 10 remaining compounds. Some ligands exhibited large differences in potency for the different pathways, suggesting that the nature of the receptor-effector complexes influences the relative affinity of the compounds for specific receptor conformations. Calculation of relative effectiveness (within pathway) and bias factors (between pathways) for each of the compounds, using an operational model of agonism, revealed a global signaling signature for all of the compounds relative to isoproterenol. Most compounds were biased toward ERK1/2 activation over the other pathways, consistent with the notion that many proximal effectors converge on this pathway. Overall, we demonstrate a higher level of ligand texture than previously anticipated, opening perspectives for the establishment of pluridimensional correlations between signaling profiles, drug classification, therapeutic efficacy, and safety.

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades.

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3'-5'-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:ßγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades.

Multiplexing of multicolor bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET) is increasingly being used to monitor protein-protein interactions and cellular events in cells. However, the ability to monitor multiple events simultaneously is limited by the spectral properties of the existing BRET partners. Taking advantage of newly developed Renilla luciferases and blue-shifted fluorescent proteins (FPs), we explored the possibility of creating novel BRET configurations using a single luciferase substrate and distinct FPs. Three new (to our knowledge) BRET assays leading to distinct color bioluminescence emission were generated and validated. The spectral properties of two of the FPs used (enhanced blue (EB) FP2 and mAmetrine) and the selection of appropriate detection filters permitted the concomitant detection of two independent BRET signals, without cross-interference, in the same cells after addition of a unique substrate for Renilla luciferase-II, coelentrazine-400a. Using individual BRET-based biosensors to monitor the interaction between G-protein-coupled receptors and G-protein subunits or activation of different G-proteins along with the production of a second messenger, we established the proof of principle that two new BRET configurations can be multiplexed to simultaneously monitor two dependent or independent cellular events. The development of this new multiplexed BRET configuration opens the way for concomitant monitoring of various independent biological processes in living cells.

Vasopressin type 2 receptor V88M mutation: molecular basis of partial and complete nephrogenic diabetes insipidus.

BACKGROUND/AIMS: Mutations in the type 2 vasopressin receptor gene (AVPR2) underlie X-linked recessive nephrogenic diabetes insipidus (NDI). Here, we report on a family with a mutation in AVPR2, c.262G>A (p.V88M). This recurrently identified mutation was previously shown to abolish AVPR2 function, yet in some affected members, urine osmolalities of up to 570 mosm/kg were observed. We detail the variable clinical phenotype and investigate its molecular basis. METHODS: Retrospective analysis of clinical data and in vitro assessment of wild-type and V88M-mutant receptors. RESULTS: Clinical data were available on 6 patients. Four of these demonstrated a substantial increase in urinary concentration after 1-desamino[8-D-arginine] vasopressin, consistent with partial NDI, while 2 did not respond. In vitro analysis revealed a reduced cell surface expression and decreased binding affinity for arginine-vasopressin of the mutant receptor, leading to blunted signaling activity. Treatment with the pharmacological chaperone SR121463 enhanced cell surface expression. CONCLUSION: The V88M mutation is associated with phenotypical diversity, which may be explained by the fact that both the expression level and the hormone-binding affinity are affected by the mutation. Our results provide a rational basis for treatment trials with vasopressin analogues in combination with pharmacologic chaperones in patients with this recurrently identified mutation.